The invention relates to highly radioactive 1,4-dihydropyridine derivatives and 1,4-dihydroquinoline derivatives labelled with .sup.125 I, their preparation by reacting 1,4-dihydropyridine esters or 1,4-dihydroquinoline esters bearing a terminal amino group on the ester group with .sup.125 I-labelled acylating compounds, and the use of the compounds separated by preparative chromatographic methods for testing medicaments or other substances with the aid of receptors (medicament screening with the aid of radio-receptor assays) and for measuring the plasma levels of calcium channel active medicaments.
A rapid, autoradiographic identification of receptor binding sites of medicaments and other substances that interact with 1,4-dihydropyridine or 1,4-dihydroquinoline binding sites (receptors), is thereby also possible.
The term "organic calcium antagonists" (A. Fleckenstein: Calcium Antagonism in Heart and Smooth Muscle, John Wiley and Sons, New York, Clinchester, Bristone, Singapore, 1983; D. J. Triggle: Calcium Antagonists: Basic Chemical and Pharmacological Aspects (p.p. 1-18) in G. B. Weiss (Editor): New Perspective on Calcium Antagonists, American Physiological Society, Bethseda, Md., USA., 1981; D. J. Triggle: Chemical Pharmacology of Calcium Antagonists (p.p. 17-38) in R. G. Rahwan and D. T. Witiak (eds.): Calcium Regulation by Calcium Antagonists. ACS Symposium Series 201. American Chemical Society, Washington, DC, 1982) covers substances used as therapeutics in man and which have the following areas of application at the present time: angina pectoris, supraventricular tachycardia, ventricular cardiac rhythm disturbances, artrial flutter and atrial fibrillation, raised blood pressure, and are mentioned as suitable for the following possible areas of application: cerebral insufficiency and vasospasm, high pulmonary pressure, bronchial asthma, premature birth, dysmenorrhoea, cardiac protection, arteriosclerosis and blocking of the release of so-called mediators (Thromboxan A.sub.2) in allergic or inflammatory disorders or disorders involving blood platelet aggregation (A. Fleckenstein in: Calcium Antagonism in Heart and Smooth Muscle, 1983 and R. A. Janis and D. J. Triggle in: New Developments in Ca.sup.2+ Channel Antagonists. J. Med. Chem. 26, 775-785, 1983).
The therapeutically used calcium antagonists and calcium antagonists in the clinical trial or development stage belong to various chemical classes. 1,4-dihydropyridines and 1,4-dihydroquinolines are far and away the most effective of the hitherto known calcium antagonists.
Even in concentrations of 10.sup.-10 -10.sup.-9 mole/liter these substances block the passage of calcium through the calcium channels of smooth muscle cell membranes (A. Fleckenstein, 1983; D. J. Triggle, 1981, 1982; R. A. Janis and D. J. Triggle, 1983). This blocking is stereoselective in the case of chiral 1,4-dihydropyridines (Towart, R., Wehinger, E. and Meyer, H. (1981): Effects of unsymmetrical ester substituted 1,4-dihydropyridine derivatives and their optical isomers on contraction of smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmacol. 317: 183-185; Towart R., Wehinger, E., Meyer, H., Kazda, S. (1982): The effects of nimodipine, its optical isomers and metabolites on isolated vascular smooth muscle. Arzneim. Forsch. Drug Res) The following publications: Glossmann, H., Ferry, D. R., Lubbecke, F., Mewes, R., Hoffman, F. (1982): Calcium channels: direct identification with radioligand binding studies. TIPS 3: 431-437; Glossman, H. and Ferry, D. R. (1983): Molecular approach to the calcium channel. Drug Development 9: 63-98; Janis, R. A., Triggle, D. J. (1983): New developments in Ca.sup.2+ channel antagonists. J. Med. Chem. 26; 775-785; Janis, R. A., Scriabine, A. (1983): Sites of action of Ca.sup.2+ channel inhibitors. Biochem. Pharmacol. (in press); Ferry, D. R., Goll, A., Glossmann, H. (1983): Differential labelling of putative skeletal muscle calcium channels by .sup.3 H-nifedipine, .sup.3 H-nitrendipine, .sup.3 H-nimodipine and .sup.3 H-PN200-110. Nauyn-Schmiedeberg's Arch. Pharmacol. 323: 276-277; Bellemann, P., Ferry, D. R., Lubbecke, F., Glossmann, H. (1981); .sup.3 H-nitrendipine, a potent calcium antagonist binds with high affinity to cardiac membranes. Arzneim-Forsch (Drug Res) 31: 2064-2067; Ferry, D. R., Glossmann, H. (1982a): Identification of putative calcium channels in skeletal muscle microsomes. FEBS Lett 148: 331-337; Ferry, D. R., Glossmann, H. (1982b): Evidence for multiple drug receptor sites within the putative calcium channel. Naunyn-Schmiedeberg's Arch. Pharmacol. 321: 80-83, describe, inter alia, in review articles the use of Tritiated (specific activity approx. 3-approx. 160 Ci/mMole) 1,4-dihydropyridines to characterise the binding sites (receptors) for these phamraceuticals. Tritiated 1,4-dihydropyridines may be used, on the basis of this knowledge of in vitro screening of new substances, to elucidate specific side effects of known medicaments or to measure the plasma level of calcium antagonists. (Examples: R. J. Gould, K. M. M. Murphy, I. J. Reynolds and S. H. Snyder: Thioridazine: Calcium channel blockade may explain peripheral side effects. American J. Psychiatrics (in press) and R. J. Gould, K. M. M. Murphy and S. H. Snyder: A simple radioreceptor assay for calcium antagonist drugs.
Life Sciences (in press)). Tritiated 1,4-dihydropyridines are also used in the autoradiographic identification of their receptors (e.g. Quirion, R. (1983): Autoradiographic localisation of a calcium channel antagonist. .sup.3 H-nitrendipine, binding site in rat brain (Neuroscience Lett 36: 267-271).
According to the present state of the art and current regulations concerning the elimination of radioactive waste, tritiated compounds are no longer regarded as optimum substances for the afore-mentioned examples of use. Disadvantages of .sup.3 H compared with, for example, .sup.125 I are the long half-life value, the relatively low specific activity and therefore reduced sensitivity, the need to use organic scintillators, the poor counting efficiency, and the long exposure times for the purposes of autoradiography.
The problems of high level radioactive labelling with .sup.125 I of substances that act for example as haptens and are used as ligands in a radioimmunoassay have been exhaustively discussed by J. E. Corrie and W. M. Hunter (Methods in Enzymology Vol. 73, Part B, Eds. J. L. Langone and H. van Vunakis, Academic Press. New York, London, Toronto, Sydney, San Francisco, 1981, p.p. 79-112). According to them, it is of paramount importance that the ligand produced is analytically effective in the field of application, that the radioactive labelling is reproducible, and that the ligand exhibits a low non-specific binding and can be stored in a stable condition for a prolonged period (days, weeks, months). There are in principle three ways of synthesizing stable .sup.125 I-labelled ligands:
1. (And the least used) Halogen exchange reactions (e.g. Science 205, p.p. 1138-1140 (1979))
2. Introduction of .sup.125 I in phenol or imidazole derivatives by means of the chloramine T method according to Greenwood, Hunter and Glover, or electrolytically or by means of lactoperoxidase (very commonly used), and
3. The introduction of radioactive .sup.125 I via a carrier molecule. With a few exceptions (e.g. aminoglycoside antibiotics, Clonazepam, Biotin, Bleomycin) this third method namely the introduction of radioactive .sup.125 I via a carrier molecule, e.g. with .sup.125 I-labelled N-succinimidyl 3-(4-hydroxyphenyl)-propionate, di-.sup.125 I-labelled methyl p-hydroxybenzimidate and .sup.125 I-labelled diazotised aniline, is described only for peptides and proteins or e.g. for heparin (J. J. Langone in: Methods in Enzymology Vol. 73, Part B, Eds. J. L. Langone and H. van Vunakis, Academic Press, New York, London, Toronto, Sydney, San Francisco, 1981, p.p. 112-127).
The basic limitation of the method follows from the absolute priority as regards the properties, demanded by J. E. Corrie and W. M. Hunter (ibid.), namely the ligand produced must not only be radioactive, but must also be analytically utilisable. It is therefore not sufficient just to introduce radioactive iodine into a molecule via one of the afore-mentioned methods, in particular method 3; the labelled product must also be recognised, e.g. in a radioimmunoassay, by an antibody directed against it, and must be able to be bound with a sufficient binding strength. The smaller the molecule to be radioactively labelled, the more difficult it becomes to satisfy the structural requirements, in particular for specific binding sites (receptors) of the medicaments, if by means of method 3 a relatively large radioactive substance alters the structure via the introduction of a carrier molecule. These difficulties explain why highly radioactive 1,4-dihydropyridines and 1,4-dihydroquinolines labelled with .sup.125 I that satisfy the necessary criteria do not yet exist.